Microtubule motor proteins are responsible for many aspects of intracellular transport and cytoplasmic dynein is a major minus end- directed motor in mammalian cells which orchestrates the movement of membranes; chromosomes and mitotic spindles. We do not understand how cytoplasmic dynein identifies and binds cargo or how this process is regulated. Several rival hypotheses have been proposed to explain cargo-binding and regulation, implicating different subunits and different receptor molecules. The goals of this proposal are to evaluate these hypotheses. The Specific Aims of this proposal are: 1) To Map Dynein Phosphorylation Sites That Regulate Cargo Binding, 2) To Define Dynein Subunits Responsible for Cargo Binding and 3) To Determine the Mechanisms of Cargo Release. In Aim 1, we will isolate cytoplasmic dynein that has been released from cargo during mitosis, identify phosphorylated subunits and map phosphorylated residues using mass spectrometry. In Aim 2, we will use recombinant dynein subunits as well as disruption of the dynein complex to identify the minimal dynein subcomplex capable of binding organelles. In Aim 3, we will test the impact of phosphorylation on subunit function by creating site-directed mutants in dynein subunits designed to mimic phosphorylated and dephosphorylated versions of the protein. These mutants will be compared to wild-type subunits in organelle binding and motility assays . These studies will test existing hypotheses of dynein targeting and regulation. Through identification of cargo binding subunits and regulatory pathways that control dynein function, this work will advance our understanding of molecular motors and provide new tools for future work.